Peptide nucleic acid incorporating a chiral backbone

ABSTRACT

A novel class of peptide nucleic acid monomers are synthesized having chirality in the backbone. Peptide nucleic acid oligomers are synthesized to incorporate these chiral monomers.

CROSS REFERENCE TO RELATED APPLICATIONS

This Application is a continuation of application Ser. No. 08/366,231filed Dec. 28. 1994 now U.S. Pat. No. 5,977,296 which is acontinuation-in-part of Ser. No. 08/108,591 filed Nov. 22, 1993 that, inturn, is a national phase application of PCT Application Ser. No.EP/01219, filed May 22, 1992, claiming priorit to Danish PatentApplications No. 986/91, filed May 24, 1991, No. 987/91 filed May 24,1991 and No. 510/92. filed Apr. 15, 1992. Each of the foregoing patentapplications are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention is directed to compounds that are not polynucleotides yetwhich bind to complementary DNA and RNA strands more strongly than thecorresponding DNA. In particular, the invention concerns peptide nucleicacids (PNAs) which are synthesized to have a chiral backbone.

BACKGROUND OF THE INVENTION

Oligonucleotides and their analogs have been developed and used inmolecular biology in certain procedures as probes, primers, linkers,adapters, and gene fragments. Modifications to oligonucleotides used inthese procedures include labeling with non isotopic labels, e.g.fluorescein, biotin, digoxigenin, alkaline phosphatase, or otherreporter molecules. Other modifications have been made to the ribosephosphate backbone to increase the nuclease stability of the resultinganalog. These modifications include use of methyl phosphonates,phosphorothioates, phosphorodithioate linkages, and 2′-O-methyl ribosesugar units. Further modifications, include modifications made tomodulate uptake and cellular distribution. Phosphorothioateoligonucleotides are presently being used as antisense agents in humanclinical trials for various disease states including use as antiviralagents. With the success of these oligonucleotides for both diagnosticand therapeutic uses, there exists an ongoing demand for improvedoligonucleotide analogs.

Oligonucleotides can interact with native DNA and RNA in several ways.One of these is duplex formation between an oligonucleotide and a singlestranded nucleic acid. The other is triplex formation between anoligonucleotide and double stranded DNA to form a triplex structure.

Peptide nucleic acids are compounds that in certain respects are similarto oligonucleotide analogs however in other very important respectstheir structure is very different. In peptide nucleic acids, thedeoxyribose backbone of oligonucleotides has been replaced with abackbone more akin to a peptide than a sugar. Each subunit, or monomer,has a naturally occurring or non naturally occurring base attached tothis backbone. One such backbone is constructed of repeating units ofN-(2-aminoethyl)glycine linked through amide bonds. Because of theradical deviation from the deoxyribose backbone, these compounds werenamed peptide nucleic acids (PNAs).

PNA binds both DNA and RNA to form PNA/DNA or PNA/RNA duplexes. Theresulting PNA/DNA or PNA/RNA duplexes are bound with greater affinitythan corresponding DNA/DNA or DNA/RNA duplexes as determined by Tm's.This high thermal stability might be attributed to the lack of chargerepulsion due to the neutral backbone in PNA. The neutral backbone ofthe PNA also results in the Tm's of PNA/DNA(RNA) duplex beingpractically independent of the salt concentration. Thus the PNA/DNAduplex interaction offers a further advantage over DNA/DNA duplexinteractions which are highly dependent on ionic strength.Homopyrimidine PNAs have been shown to bind complementary DNA or RNAforming (PNA)2/DNA(RNA) triplexes of high thermal stability (see, e.g.,Egholm, et al., Science, 1991, 254, 1497; Egholm, et al., J. Am. Chem.Soc., 1992, 114, 1895; Egholm, et al., J. Am. Chem. Soc., 1992, 114,9677).

In addition to increased affinity, PNA has also been shown to bind toDNA with increased specificity. When a PNA/DNA duplex mismatch is meltedrelative to the DNA/DNA duplex there is seen an 8 to 20° C. drop in theTm. This magnitude of a drop in Tm is not seen with the correspondingDNA/DNA duplex with a mismatch present.

The binding of a PNA strand to a DNA or RNA strand can occur in one oftwo orientations. The orientation is said to be anti-parallel when theDNA or RNA strand in a 5′ to 3′ orientation binds to the complementaryPNA strand such that the carboxyl end of the PNA is directed towards the5′ end of the DNA or RNA and amino end of the PNA is directed towardsthe 3′ end of the DNA or RNA. In the parallel orientation the carboxylend and amino end of the PNA are just the reverse with respect to the5′-3′ direction of the DNA or RNA.

PNAs bind to both single stranded DNA and double stranded DNA. As notedabove, in binding to double stranded DNA it has been observed that twostrands of PNA can bind to the DNA. While PNA/DNA duplexes are stable inthe antiparallel configuration, it was previously believed that theparallel orientation is preferred for (PNA)₂/DNA triplexes.

The binding of two single stranded pyrimidine PNAs to a double strandedDNA has been shown to take place via strand displacement, rather thanconventional triple helix formation as observed with triplexingoligonucleotides. When PNAs strand invade double stranded DNA, onestrand of the DNA is displaced and forms a loop on the side of thePNA₂/DNA complex area. The other strand of the DNA is locked up in the(PNA)₂/DNA triplex structure. The loop area (alternately referenced as aD loop) being single stranded, is susceptible to cleavage by enzymesthat can cleave single stranded DNA.

A further advantage of PNA compared to oligonucleotides is that theirpolyamide backbone (having appropriate nucleobases or other side chaingroups attached thereto) is not recognized by either nucleases orproteases and are not cleaved. As a result PNAs are resistant todegradation by enzymes unlike nucleic acids and peptides.

Because of their properties, PNAs are known to be useful in a number ofdifferent areas. Since PNAs having stronger binding and greaterspecificity than oligonucleotides, they are used as probes in cloning,blotting procedures, and in applications such as fluorescence in situhybridization (FISH). Homopyrimidine PNAs are used for stranddisplacement in homopurine targets. The restriction sites that overlapwith or are adjacent to the D-loop will not be cleaved by restrictionenzymes. Also, the local triplex inhibits gene transcription. Thus inbinding of PNAs to specific restriction sites within a DNA fragment,cleavage at those sites can be inhibited. Advantage can be taken of thisin cloning and subcloning procedures. Labeled PNAs are also used todirectly map DNA molecules. In effecting this, PNA molecules having afluorescent label are hybridized to complementary sequences in duplexDNA using strand invasion.

PNAs have further been used to detect point mutations in PCR-basedassays (PCR clamping). PCR clamping uses PNA to detect point mutationsin a PCR-based assay, e.g. the distinction between a common wild typeallele and a mutant allele, in a segment of DNA under investigation. APNA oligomer complementary to the wild type sequence is synthesized. ThePCR reaction mixture contains this PNA and two DNA primers, one of whichis complementary to the mutant sequence. The wild type PNA oligomer andthe DNA primer compete for hybridization to the target. Hybridization ofthe DNA primer and subsequent amplification will only occur if thetarget is a mutant allele. With this method, one can determine thepresence and exact identity of a mutant.

Considerable research is being directed to the application ofoligonucleotides and oligonucleotide analogs that bind complementary DNAand RNA strands for use as diagnostics, research reagents and potentialtherapeutics. PCT/EP/01219 describes novel peptide nucleic acid (PNA)compounds which bind complementary DNA and RNA more tightly than thecorresponding DNA. Because of these binding properties as well as theirstability, such PNA compounds find many uses in diagnostics andreasearch reagents uses associated with both DNA and RNA. Withcomplementary DNA and RNA they can form double-stranded, helicalstructures mimicking double-stranded DNA, and they are capable of beingderivatized to bear pendant groups to further enhance or modulate theirbinding, cellular uptake, or other activity.

PNA compounds having cyclic backbones have been described byPCT/US93/05110. These compounds are believed to have increasedconformational restriction which lends to increased binding affinity andspecificity. However, these monomers, and oligomers comprising thesemonomers, are racemic mixtures which result in reduced bindingspecificity. Thus, compositions comprising single enantiomeric speciesand methods of making the same, are greatly desired.

OBJECTS OF THE INVENTION

It is an object of the invention to provide PNAs having a chiralbackbone.

It is a further object of the present invention to provide PNA oligomershaving at least one chiral PNA monomer.

It is yet a furtherlobject of the present invention to provide methodsof producing these novel compounds.

These and other objects will become apparent from the followingdescription and accompanying claims.

SUMMARY OF THE INVENTION

The present invention is directed to novel peptide nucleic acid monomersthat contain an aliphatic cyclic structure in the backbone resulting ina chiral backbone. The present invention is also directed to peptidenucleic acid oligomers that incorporate these monomers.

Oligomers of the present invention are useful as research reagents andas diagostic tools. Compounds of the present invention can be used todetect point mutations in a sample of DNA of interest. Otherapplications include enabling PCR amplification of a mutant gene DNAwhile the wild type is suppressed by hybridization to a chiral PNA. Manyapplications of the present invention will become evident to thoseskilled in the art.

Compounds of the invention include peptide nucleic acid monomers of theformula:

wherein:

B is a naturally or non-naturally ocurring nucleobase;

n is 0, 1, 2, or 3; and

at least one of Cα or Cβ is in the S configuration.

In a preferred embodiment of the invention both Cα and Cβ are in the Sconfiguration.

In a further preferred embodiment of the invention B is adenine,cytosine, guanine, thymine, or uracil.

In still another preferred embodiment of the invention n is 2.

Further in accordance with this invention there are provided monomers ofthe formula:

wherein:

B is a naturally or non-naturally ocurring nucleobase; and

at least one of Cα or Cβ is in the S configuration.

In a preferred embodiment of the invention Cα and Cβ are in the Sconfiguration.

In a further preferred embodiment of the invention B is adenine,cytosine, guanine, thymine, or uracil.

Compounds of the invention further include peptide nucleic acidoligomers complementary to a target molecule. The oligomers comprise atleast one peptide nucleic acid monomer having a (2-aminoethyl)glycinebackbone with a chiral center in the ethyl portion of the backbone. Themonomer is incorporated into peptide nucleic acid oligomers of thepresent invention at a position corresponding to a region of variabilityin the target molecule.

Further in accordance with the present invention there are providedoligomers comprising at least two peptide nucleic acid monomers, atleast one of said peptide nucleic acid monomers having the structure:

wherein:

B is a naturally or non-naturally ocurring nucleobase;

at least one of Cα or Cβ is in the S configuration;

Q is —OH, a carbonyl protecting group, or a covalent bond;

I is H, an amino protecting group, or a covalent bond; and

n is 0, 1, 2, or 3.

In a preferred embodiment of the invention Cα and Cβ are in the Sconfiguration.

In a further preferred embodiment of the invention B is adenine,cytosine, guanine, thymine, or uracil.

In yet a further preferred embodiment of the invention n is 2.

BRIEF DESCRIPTION OF THE FIGURES

FIG. I illustrates a synthetic scheme according to the invention anddiscussed in Examples 1-8.

FIG. II illustrates the circular dichroism spectra of chiral PNAshybridized to their complementary DNAs.

DESCRIPTION OF PREFERRED EMBODIMENTS

Specific sequence recognition of DNA or RNA is of increasing importancefor the development of biological probes and new reagents for use inresearch (Uhlmann, E., Peyman, A., Chem. Rev., 1990, 90, 544, andHelene, C., Toulme, J. J., Biochim. Biophys. Acta., 1990, 1049, 99).Peptide nucleic acid (PNA), an achiral analog of DNA where thenucleobases or nucleobase analogs are attached to a(2-aminoethyl)-glycine backbone through a methylene carbonyl linker haveproperties making them well suited for use as biological probes andother applications. PNA have shown strong binding affinity andspecificity to complementary DNA, sequence specific inhibition of DNArestriction enzyme cleavage and site specific in vitro inhibition oftranslation (Egholm, M., et.al., Chem. Soc., Chem. Commun., 1993, 800;Egholm, M., et.al., Nature, 1993, 365, 566; Nielsen, M., et.al. Nucl.Acids Res., 1993, 21, 197; and Hanvey, J. C., et.al., Science, 1992,258, 1481). Modifications of PNA include extended backbones (Hyrup, B.,et.al. Chem. Soc., Chem. Commun., 1993, 518), extended linkers betweenthe backbone and the nucleobase, reversal of the amido bond (Lagriffoul,P. H., et.al., Biomed. Chem. Lett., 1994, 4, 1081), and the use of achiral backbone based on alanine (Dueholm, K. L, et.al., BioMed. Chem.Lett., 1994, 4, 1077).

This invention is directed to a modification of PNA that has increasedspecificity while maintaining comparable affinity. This is acheived bythe introduction of chirality into the backbone through an aliphaticcyclic structure incorporated to include the Cα and Cβ of the2-aminoethyl portion of the backbone. The resulting monomer hasincreased conformational restriction. The added aliphatic cyclic ringsystem is also expected to increase the lipophilicity of the monomer.Thus, this invention is directed to novel PNA molecules having a chiralbackbone. Peptide nucleic acid monomers of the present invention havethe formula:

wherein:

B is a naturally or non-naturally ocurring nucleobase;

n is 0, 1, 2, or 3;

and at least one of Cα or Cβ is in the S configuration.

In some embodiments of the present invention B is a naturally occuringDNA nucleobase such as adenine, cytosine, guanine or thymine or othernaturally occurring nucleobases (e.g., inosine, uracil, 5-methylcytosineor thiouracil). In still other embodiments of the present invention B isa non-naturally occuring nucleobase (e.g., bromothymine, azaadenines orazaguanines, etc.).

The Cα and Cβ of the 2-aminoethyl portion of the 2-aminoethylglycinebackbone are synthesized to be part of an aliphatic cyclic structure.This aliphatic cyclic structure may be a 4, 5, 6 or 7 membered ring. Inpreferred embodiments the aliphatic cyclic structure is a 6 memberedring.

The use of optically active reagents permits the synthesis of pure SS,RR, SR, and RS isomers. The SS isomer is preferred in some embodimentsof the present invention.

Monomers having a chiral backbone are prepared using (1,2)-diaminocyclohexane, which is available as the cis, or the trans isomer.The cis-(l,2)-diaminocyclohexane is a meso compound. Use of such mesocompound requires purification of a racemic mixture. Thetrans-(1,2)-diaminocyclohexane is commercially available inenantiomerically pure form, making it well suited for monomers ofpredetermined chirality about both the Cα and the Cβ of the 2-aminoethylportion of the backbone.

In FIG. I the diamine is protected at one of the amino groups withdi-t-butylpyrocarbonate (Boc₂O), followed by N-alkylation with methylbromoacetate to give the chiral backbone. Coupling of a carboxymethylfunctionalized nucleobase (e.g. a 1-carboxymethyl pyrimidine, or a9-carboxymethyl purine) or nucleobase analog with the chiral backboneusing DCC/DhbtOH followed by basic hydrolysis will give the desiredmonomer containing a chiral backbone. In this manner the SS monomer,N-(2S-Boc-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine and the RRmonomer, N-(2R-Boc-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl)-glycinewere synthesized. The RS and the SR isomers can be synthesized using theCis-(1,2)-diaminocyclohexane and separating the racemic mixture thatresults. Separation can be achieved, for example, by liquidchromatography.

PNA oligomers comprising at least one chiral monomer are prepared inaccordance with methods known to those skilled in the art. Establishedmethods for the stepwise or fragmentwise solid-phase assembly of aminoacids into peptides normally employ a beaded matrix of slightlycross-linked styrene-divinylbenzene copolymer, the cross-linkedcopolymer having been formed by the pearl polymerization of styrenemonomer to which has been added a mixture of divinylbenzenes. A level of1-2% cross-linking is usually employed. Such a matrix also can be usedin solid-phase PNA synthesis in accordance with the present invention.Preferably, the PNA oligomer is prepared to be complementary to a targetmolecule, i.e. at least a portion of the PNA oligomer has the ability tohybridize due to Watson-Crick base pair attraction to the targetmolecule.

The thermal stability of homopyrimidine PNA/DNA and homopyrimidinePNA*/DNA wherein PNA* denotes a PNA oligomer containing one chiral (SSor RR) monomer was studied to determine the effects of the chiralmonomer on the Tm. It has been previously shown that a homothymine PNAdecamer forms a very stable 2:1 complex with its complementary DNA.Introduction of one mismatch in the DNA strand resulted in a significantdestabilization of the PNA/DNA complex. When the SS isomerH-TTTTT**TTTTT-Lys-NH₂ (SEQ ID NO:4) (where T** denotes the SSmonomer,N-(2S-Boc-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine)was hybridized with the DNA 10mer A₁₀, the Tm was comparable to that ofthe PNA(H-TTTTT**TTTTT-Lys-NH₂) (SEQ ID NO:4)/DNA complex. Introductionof a mismatch in the DNA 10mer corresponding to the position of thechiral SS monomer in the chiral PNA 10mer resulted in the samedestabilization as the PNA without a chiral monomer present. The resultsof this study show that the 10mer containing the SS isomer showscomparable binding affinity and equivalent specificity when compared tothe 10mer PNA without the SS isomer.

When the same thermal stability studies were performed on the RR isomer,N-(2R-Boc-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl) -glycine, there wasseen poor binding affinity as well as poor specificity.

Circular dichroism and linear dichroism studies have shown thathomopyrimidine PNA/DNA triplexes have a very similar structure to thatof the conventional DNA/DNA triplex (Kim, S. K., et.al., J. Am. Chem.Soc., 1993, 115, 6477). Figure II shows the CD spectra of the SS and theRR containing 10mer PNAs hybridized to their complementary DNAs. The SSand RR containing 10mers alone have a weak CD due to the presence of thechiral monomer and the L-Lys residue. Upon hybridization, a strong CDresponse, very much like that of the normal PNA/DNA complex, arose. Inagreement with Tm studies, the SS containing 10mer gave a stronger CDspectra that the RR containing 10mer. Although the two chiral containing10mers are of different configuration, the complexes (SS 10mer and RR10mer) gave rise to CD spectra that are not mirror-image. Thus, theincorporation of one chiral monomer did not disrupt the right-handedhelical structure of the PNA/DNA complex.

The thermal stability of mixed PNA/DNA and mixed PNA*/DNA sequenceswherein PNA* denotes a PNA oligomer containing one chiral (SS or RR)monomer was studied to determine the effects of the chiral monomer onthe Tm. As illustrated in Example 28, when bound to an antiparallel DNAthe oligomer containing the S,S-isomer showed comparable Tm to that ofthe unmodified PNA.

The thermal stability of a chiral PNA in a mixed sequence wasdetermined. The PNAs H-GT*AGAT*CACT*-Lys-NH₂ SEQ ID NO:13,GT**AGAT**CACT**Lys-NH₂, SEQ ID NO:14, and GTAGATCACT-Lys-NH₂ SEQ IDNO:15 wherein ** denotes an SS monomer and a * denotes an RR monomer,were synthesized as per the general procedures of Example 25. Two DNAoligonucleotides were synthesized as per known-published proceduresantiparallel to SEQ ID NO:15. These two DNA oligonucleotides differ inthe base at position 6 such that one is complementary and the other is asingle base mismatch. The mismatch ocurrs at a position that binds witha chiral position in the PNA oligomer. Each of the PNA oligomers e.g.the chiral PNA oligomers (R,R, and S,S) and the unmodified PNA oligomerwere hybridized with each of the two DNA oligomers. The relative bindingspecificity was measured using the methods and apparatus of Example 28.The results show that the PNA 10mer having an SS isomer shows greaterspecificity that the unmodified PNA 10mer (23° C. versus 10° C.).

Oligomers of the present invention are useful as research reagents andas diagostic tools. PNAs have been used in studies to discriminatebetween fully complementary and single base mismatch targets (Orum, H.,et.al., Nucleic Acids Research, 1993, 21, 5332-5336). The methodutilizes the properties of PNA e.g. higher thermal stability, greaterspecificity when bound to complementary nucleic acid sequences than thecorresponding deoxyribooligonucleotides and that PNAs. are notrecognized by DNA polymerase as primers. A PNA/DNA complex caneffectively block the formation of a PCR product when the PNA istargeted against the PCR primer site. This method is effective inblocking target sequences when two target sequences in the same assaydiffer by only one base pair. Compounds of the present invention havinggreater specificity than normal PNA are well suited for use indiagnostic assays of this type. In preferred embodiments, it ispreferred that at least one PNA monomer having a chiral center in theethyl portion of the monomer is incorporated into the PNA oligomer atthe site where a mismatch (i.e. variability of the target molecule) isexpected or known to occur.

PNA oligomers having at least one chiral monomer are easily tagged withfluorescein or rhodamine using an aminohexanoic linking moiety. Thesetagged PNA oligomers are well suited for use as probes for a section ofDNA of interest. Many other types of labeling reagents and linkingmoieties are amenable to the present invention. Many applications of thepresent invention will be evident to those skilled in the art.

The following examples are illustrative but are not meant to be limitingof the present invention.

EXAMPLE 1

(1S,2S)-1-(N-t-butyloxycarbonylamino)-2-minocyclohexane (1)

To a cooled solution of (1S,2S)-diaminocyclohexane (5 ml; 41.6 mmol) inCH₂Cl₂ (25 ml) was added a solution of di-t-butyl dicarbonate (3.03 g;13.9 mmol) in CH₂Cl₂ (25 ml) over a period of 30 mins. The reactionmixture was stirred overnight at room temperature. Water (20 ml) andCH₂Cl₂ (25 ml) were added in order to dissolve the precipitate. Afterseparation of the two phases, the organic phase was concentrated underreduced pressure and the residue was dissolved in ether (25 ml) andwater (25 ml). The mixture was acidified to pH 5 with HCl 4 N and thebis-protected diamnine was extracted with ether (3×25 ml). The aqueousphase was adjusted to 10.5 with NAOH 2 N and extracted with ethylacetate(6×30 ml). The organic phase was dried over sodium sulfate, filtered andevaporated under reduced pressure to yield the title-compound (2.3 g,73% based on Boc₂O).

MP:109-111° C.; NMR¹H(dmso d6) ppm: 1.0-1.3 (mn, 4H. 2CH₂ cycl) ; 1.45(s, 9 H, t-Boc) ; 1.6 (m, 2 H, CH₂ cycl); 1.85 (m, 2 H, CH₂ cycl) ; 2.4(dt, 1 H, CHN) ; 2.9 (m, 1 H, CHN) ; 6.6 (m, 1 H, NH carbamate); NMR ¹³C(dmso d6) ppm: 155.6 (carbamate); 77.4 (t-Boc) ; 57.0, 53.8, 34.4, 32.2,25.8, 24.8 (C cycl); 28.4 (t-Boc).

EXAMPLE 2

N-(2S-Boc-aminocyclohex-1S-yl)-glycine, methyl ester (2)

To a cooled suspension of 1 (2 g, 9.35 mmol) and potassium carbonate(3.87 g, 28.05 mnmol) in DMF (15 ml) was added a solution of methylbromoacetate (0.9 ml, 9.35 mmol) in DMF (5 ml) over a period of 5 min.After one hour at 0° C. the salts were filtered and washed with DMF andCH₂Cl₂. The filtrate was evaporated under reduced pressure and theresidue was purified by chromatography on silica gel (eluentethylacetate). Yield: 1.9 g (70%).

mp:68-70° C.; NMR¹H (dmso d6) ppm: 1.0-1.3 (m,4 H,2CH₂ cycl); 1.45 (s, 9H, t-Boc); 1.4-2.0 (m,5 H,2CH₂ cycl+NH); 2.3 (dt, 1 H, CHN) ; 3.1 (m, 1H, CHN) ; 3.4 (dt, 2 H, CH₂COO) ; 3.7 (s, 3 H, COOCH₃); 6.7 (m, 1 H, NHcarbamate); NMR ¹³C (dmso d6) ppm: 172.9 (ester); 155.5 (carbamate);77.5 (t-Boc); 59.7, 47.6 (CH₂COOCH₃); 53.7, 51.4, 32.1, 31.1, 24.6, 24.2(C cycl); 28.3 (t-Boc); MSFAB+: 287.0 (M+1).

EXAMPLE 3

N-(2S-Boc-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl) -glycine, methylester (3)

To a solution of 2 (1.5 g, 5.24 mmol), thymine acetic acid (0.96 g; 5.22mmol) and DhbtOH (0.85 g, 5.2 mmol) in DMF (15 ml) and CH₂Cl₂ (15 ml)was added DCC (1.08 g, 5.24 mmol). After 4 hrs at room temperature, DCUwas filtered and washed with CH₂Cl₂ (100 ml). The filtrate was washedwith NaHCO₃ 1 M (3×40 ml), KHSO₄ 1 M (2×40 ml), H₂O (40 ml). The organicphase was dried over sodium sulfate and filtered. Petroleum ether (100ml) was added. After 48 h at 0° C., the title compound was collected byfiltration. Yield: 1.7 g (72%).

mp: 205-207° C.; NMR ¹ H (dmso d6) ppm: 1.2-2.0 (m, CH₂ cycl); 1.45 (s,t-Boc); 1.9 (CH₃ thymine); 3.7 (S, COOCH₃); 3.7 (dd, CH₂COO); 4.8 (dd,CH₂-T); 6.95 (m, NH carbamate); 7.2 (s, H-C═C-Me); 11.35 (s, NH imide);NMR ¹³C (dmso d6) ppm: 169.7, 167.1, 164.3, 154.9, 150.9 (C═O); 141.5,108.2 (C═C); 77.9 (t-Boc); 59.8 47.5 (CH₂COOCH₃); 53.7, 51.4, 32.1,31.1, 24.6, 24.2 (C cycl); 28.2 (t-Boc); 11.9 (CH₃ thymine); MS FAB+:453.3 (M+1); 353.3 (M+1 -t-Boc).

EXAMPLE 4

N-2-(2S-Boc-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine (4)

The monomer ester 3 (1.5 g, 3.3 mmol) was suspended in THF (15 ml) and asolution of LiOH 0.5 M (15 ml; 7.5 mmol) was added as well as water (5ml). After 45 minutes at room temperature, water (10 ml) was added andthe mixture washed with ethylacetate (2×10 ml). The aqueous phase wasacidified to pH 3 and extracted with ethylacetate (4×120 ml). Theorganic phase was dried over sodium sulfate and evaporated under reducedpressure. Yield: 1.36 g (94%).

NMR ¹H (dmso d6) ppm: 1.2-2.0 (m, CH₂ cycl); 1.45 (s, t-Boc); 1.9 (CH₃thymine); 3.9 (dd, CH₂COO); 4.8 (dd, CH₂-T); 6.95 (m, NH carbamate); 7.2(s, H-C═C-Me); 11.35 (s, NH imide); 12.4 (m, COOH); NMR ¹³C (dmso d6)ppm: 170.3, 166.8, 164.3, 155.0, 150.9 (C═O); 141.5, 108.1 (C═C); 77.9,28.2 (t-Boc); 59.7, 49.9, 47.9, 44.2, 32.1, 29.7, 24.4, 24.3, (Ccycl+2CH₂); 11.9 (CH₃ thymine); MS FAB+439.2 (M+1) ; 339.1 (M+1-t-Boc).

EXAMPLE 5

N-4-Cbz cytosine (5)

Over a period of about 1 h, Cbz chloride (52 ml; 0.36 mol) was addeddropwise to a suspension of cytosine (8, 20.0 g;0.18 mol) in drypyridine (1000 ml) at 0° C. under nitrogen in oven-dried equipment. Thesolution then was stirred overnight, after which the pyridine suspensionwas evaporated to dryness, in vacuo. Water (200 ml) and 4 N hydrochloricacid were added to reach pH ˜1. The resulting white precipitate wasfiltered off, washed with water and partially dried by air suction. Thestill-wet precipitate was boiled with absolute ethanol (500 ml) for 10min, cooled to 0° C., filtered, washed thoroughly with ether, and dried,in vacuo. Yield 24.7 g (54%). M.p.>250° C. Anal. for C₁₂H₁₁N₃O₃.Found(calc.); C: 58.59 (58.77); H: 4.55 (4.52); N: 17.17 (17.13). No NMRspectra were recorded since it was not possible to get the productdissolved.

EXAMPLE 6

N-4-Cbz-N-1-carboxymethyl cytosine (6)

In a three necked round bottomed flask equipped with mechanical stirringand nitrogen coverage was placed methyl bromacetate (7.82 ml; 82.6 mmol)and a suspension of N-4-Cbz cytosine (5, 21.0 g; 82.6 mmol) andpotassium carbonate (11.4 g;82.6 mmol) in dry DMF (900 ml). The mixturewas stirred vigorously overnight, filtered, and evaporated to dryness,in vacuo. Water (300 ml) and 4 N hydrochloric acid (10 ml) were added,the mixture was stirred for 15 minutes at 0° C., filtered, and washedwith water (2×75 ml). The isolated precipitate was treated with water(120 ml), 2 N sodium hydroxide (60 ml), stirred for 30 min, filtered,cooled to 0° C., and 4 N hydrochloric acid (35 ml) was added. The titlecompound was isolated by filtration, washed thoroughly with water,recrystallized from methanol (1000 ml) and washed thoroughly with ether.This afforded 7.70 g (31%) of pure compound. The mother liquor from therecrystallization was reduced to a volume of 200 ml and cooled to 0° C.This afforded an additional 2.30 g of a material that was pure by tlcbut had a reddish color. M.p. 266-274° C. Anal. for C₁₄ H₁₃N₃O₅.Found(calc.); C: 55.41 (55.45); H: 4.23 (4.32); N: 14.04 (13.86). ¹H-NMR(90 MHz; DMSO-d₆): 8.02 ppm (d,J=7.32 Hz,1 H,H-6); 7.39 (s,5 H,Ph); 7.01(d,J=7.32 Hz,1 H,H-5); 5.19 (s,2 H,PhCH₂-); 4.52 (s,2 H).

EXAMPLE 7

N-4-Cbz-N-1-carboxymethyl cytosine pentafluorophenyl ester (7)

N-4-Cbz-N-1-carboxymethyl-cytosine (6, 4.00 g; 13.2 mmol) andpentafluorophenol (2.67 g; 14.5 mmol) were mixed with DMF (70 ml),cooled to 0° C. with ice-water, and DCC (3.27 g; 15.8 mmol) was added.The ice bath was removed after 3 min and the mixture was stirred for 3 hat room temperature. The precipitated DCU was removed by filtration,washed with DMF, and the filtrate was evaporated to dryness, in vacuo(0.2 mmHg). The solid residue was treated with methylene chloride (250ml), stirred vigorously for 15 min, filtered, washed twice with dilutedsodium hydrogen carbonate and once with saturated sodium chloride, driedover magnesium sulfate, and evaporated to dryness, in vacuo. The solidresidue was recrystallized from 2-propanol (150 ml) and the crystalswere washed thoroughly with ether. Yield 3.40 g (55%). M.p. 241-245° C.Anal. for C₂₀H₁₂N₃F₅O₅. Found(calc.); C: 51.56 (51.18); H: 2.77 (2.58);N: 9.24 (8.95) .¹H-NMR (90 MHz; CDCl₃): 7.66 ppm (d,J=7.63 Hz,1 H,H-6);7.37 (s,5 H,Ph); 7.31 (d,J=7.63 Hz,1 H,H-5); 5.21 (s,2 H,PhCH₂-); 4.97(s,2 H,NCH₂-). FAB-MS: 470 (M+1)

EXAMPLE 8

N-2-(2S-Boc-aminocyclohex-1S-yl)-N-(cytosin-1-ylacetyl) -glycine (8)

The title compound is synthesized as per the procedures of Examples 3and 4 using the product from Example 7.

EXAMPLE 9

9-Carboxymethyl adenine ethyl ester (18)

Adenine (10.0 g, 74 mmol) and potassium carbonate (10.29 g, 74.0 mmol)were suspended in DMF and ethyl bromoacetate (8.24 ml, 74 mmol) wasadded. The suspension was stirred for 2.5 h under nitrogen at roomtemperature and then filtered. The solid residue was washed three timeswith DMF (10 ml). The combined filtrate was evaporated to dryness, invacuo. The yellow-orange solid material was poured into water (200 ml)and 4 N HCl was added to pH≈6. After stirring at 0° C. for 10 min, thesolid was filtered off, washed with water, and recrystallized from 96%ethanol (150 ml). The title compound was isolated by filtration andwashed thoroughly with ether. Yield 3.4 g (209). M.p. 215.5-220° C.Anal. for C₉H₁₁N₅O₂ found(calc.): C: 48.86 (48.65); H: 5.01 (4.91 ); N:31.66 (31.42 ). ¹H-NMR (250 MHz; DMSO-d₆) : (s, 2 H, H-2 & H-8), 7.25(b. s., 2 H, NH₂) , 5.06 (s, 2 H, NCH₂), 4.17 (q, 2 H, J=7.11 Hz, OCH₂)and 1.21 (t, 3 H, J=7.13 Hz, NCH₂). ¹³C-NMR. 152.70, 141.30, 61.41,43.97 and 14.07. FAB-MS. 222 (MH+). IR: Frequency in cm⁻¹ (intensity).3855 (54.3), 3274 (10.4), 3246 (14.0), 3117 (5.3), 2989 (22.3), 2940(33.9), 2876 (43.4), 2753 (49.0), 2346 (56.1), 2106 (57.1), 1899 (55.7),1762 (14.2), 1742 (14.2), 1742 (1.0), 1671 (1.8), 1644 (10.9), 1606(0.6), 1582 (7.1), 1522 (43.8), 1477 (7.2), 1445 (35.8) and 1422 (8.6).The position of alkylation was verified by X-ray crystallography oncrystals, which were obtained by recrystallization from 96% ethanol.

Alternatively, 9-carboxymethyl adenine ethyl ester 9, can be prepared bythe following procedure. To a suspension of adenine (50.0 g, 0.37 mol)in DMF (1100 ml) in 2 L three-necked flask equipped with a nitrogeninlet, a mechanical stirrer and a dropping funnel was added 16.4 g(0.407 mol) haxane washed sodium hydride- mineral oil dispersion. Themixture was stirred vigorously for 2 hours, then ethyl bromacetate (75ml, 0.67 mol) was added dropwise over the course of 3 hours. The mixturewas stirred for one additional hour, whereafter tlc indicated completeconversion of adenine. The mixture was evaporated to dryness at 1 mmHgand water (500 ml) was added to the oily residue which causedcrystallization of the title compound. the solid was recrystallized from60% ethanol (600 ml). Yield after drying 53.7 (65.6%). HPLC (215 nm)purity >99.5%.

EXAMPLE 10

N-6-Cbz-9-carboxymethyl adenine ethyl ester (10)

9-Carboxymethyladenine ethyl ester (9, 3.40 g, 15.4 mmol) was dissolvedin dry DMF (50 ml) by gentle heating, cooled to 20° C., and added to asolution of N-ethyl-Cbzimidazole tetrafluoroborate (62 mmol) inmethylene chloride (50 ml) over a period of 15 min with ice-cooling.Some precipitation was observed. The ice bath was removed and thesolution was stirred overnight. The reaction mixture was treated withsaturated sodium hydrogen carbonate (100 ml). After stirring for 10 min,the phases were separated and the organic phase was washed successivelywith one volume of water, dilute potassium hydrogen sulfate (twice), andwith saturated sodium chloride. The solution was dried over magnesiumsulfate and evaporated to dryness, in vacuo, which afforded 11 g of anoily material. The material was dissolved in methylene chloride (25 ml),cooled to 0° C., and precipitated with petroleum ether (50 ml). Thisprocedure was repeated once to give 3.45 g (63%) of the title compound.M.p. 132-35° C. Analysis for C₁₇H₁₇N₅O₄ found (calc.): C: 56.95 (57.46);H: 4.71 (4.82); N: 19.35 (19.71). ¹H-NMR (250 MHz; CDCl₃) : 8.77 (s, 1H, H-2 or H-8) ; 7.99 (s, 1 H, H-2 or H-8) ; 7.45-7.26 (m, 5 H, Ph) ;5.31 (s, 2 H, N-CH₂) ; 4.96 (s, 2 H, Ph-CH₂); 4.27 (q, 2 H, J=7.15 Hz,CH₂CH₃) and 1.30 (t, 3 H, J=7.15 Hz, CH₂CH₃). ¹³C-NMR: 153.09; 143.11;128.66; 67.84; 62.51; 44.24 and 14.09. FAB-MS: 356 (MH+) and 312(MH+-CO₂). IR: frequency in cm⁻¹ (intensity). 3423 (52.1); 3182 (52.8);3115 (52.1); 3031 (47.9); 2981 (38.6); 1747 (1.1); 1617 (4.8); 15.87(8.4); 1552 (25.2); 1511 (45.2); 1492 (37.9); 1465 (14.0) and 1413(37.3).

EXAMPLE 11

N-6-Cbz-9-carboxymethyl adenine (11)

N-6-Cbz-9-carboxymethyladenine ethyl ester (10, 3.20 g; 9.01 mmol) wasmixed with methanol (50 ml) cooled to 0° C. Sodium Hydroxide Solution(50 ml; 2 N) was added, whereby the material quickly dissolved. After 30amin at 0° C., the alkaline solution was washed with methylene chloride(2×50 ml). The aqueous solution was brought to pH 1.0 with 4 N HCl at 0°C., whereby the title compound precipitated. The yield after filtration,washing with water, and drying was 3.08 g (104%). The product containedsalt and elemental analysis reflected that. Anal. for C₁₅H₁₃N₅O₄found(calc.): C: 46.32 (55.05 ); H: 4.24 (4.00); N: 18.10 (21.40) andC/N: 2.57 (2.56). ¹H-NMR(250 MHz; DMSO-d₆): 8.70 (s, 2 H, H-2 and H-8);7.50-7.35 (m, 5 H, Ph); 5.27 (s, 2 H, N-CH₂); and 5.15 (s, 2 H, Ph-CH₂).¹³C-NMR. 168.77, 152.54, 151.36, 148.75, 145.13, 128.51, 128.17,127.98,66.76 and 44.67.IR (KBr) 3484 (18.3); 3109 (15.9); 3087 (15.0); 2966(17.1); 2927 (19.9); 2383 (53.8); 1960 (62.7); 1739 (2.5); 1688 (5.2);1655 (0.9); 1594 (11.7); 1560 (12.3); 1530 (26.3); 1499 (30.5); 1475(10.4); 1455 (14.0); 1429 (24.5) and 1411 (23.6). FAB-MS: 328 (MH+) and284 (MH+-CO₂). HPLC (215 nm, 260 nm) in system 1: 15.18 min, minorimpurities all less than 2%.

EXAMPLE 12

N-2-(2S-Boc-aminocyclohex-1S-yl)-N-(adenin-1-ylacetyl) -glycine (8)

The title compound is synthesized as per the procedures of Example 3 and4 using the product from Example 11.

EXAMPLE 13

2-Amino-6-chloro-9-carboxymethylpurine (13)

To a suspension of 2-amino-6-chloropurine (5.02 g; 29.6 mmol) andpotassium carbonate (12.91 g; 93.5 mmol) in DMF (50 ml) was addedbromoacetic acid (4.70 g; 22.8 mmol). The mixture was stirred vigorouslyfor 20 h. under nitrogen. Water (150 ml) was added and the solution wasfiltered through Celite to give a clear yellow solution. The solutionwas acidified to a pH of 3 with 4 N hydrochloric acid. The precipitatewas filtered and dried, in vacuo, over an appropriate drying agent.Yield (3.02 g; 44.8%). ¹H-NMR(DMSO-d6): d=4.88 ppm (s,2 H); 6.95 (s,2H); 8.10 (s,1 H).

EXAMPLE 14

2-Amino-6-benzyloxy-9-carboxymethylpurine (14)

Sodium (2.0 g; 87.0 mmol) was dissolved in benzyl alcohol (20 ml) andheated to 130° C. for 2 h. After cooling to 0° C., a solution of2-amino-6-chloro-9-carboxymethylpurine (13, 4.05 g; 18.0 mmol) in DMF(85 ml) was slowly added, and the resulting suspension stirred overnightat 20° C. Sodium hydroxide solution (1 N, 100 ml) was added and theclear solution was washed with ethyl acetate (3×100 ml). The water phasethen was acidified to a pH of 3 with 4 N hydrochloric acid. Theprecipitate was taken up in ethyl acetate (200 ml), and the water phasewas extracted with ethyl acetate (2×100 ml). The combined organic phaseswere washed with saturated sodium chloride solution (2×75 ml), driedwith anhydrous sodium sulfate, and taken to dryness by evaporation, invacuo. The residue was recrystallized from ethanol (300 ml). Yield afterdrying, in vacuo, over an appropriate drying agent: 2.76 g (52%). M.p.159-65° C. Anal. (calc., found) C(56.18; 55.97), H(4.38; 4.32), N(23.4;23.10). ¹H-NMR (DMSO-d₆): 4.82 ppm.(s,2 H); 5.51 (s,2 H); 6.45 (s,2 H);7.45 (m,5 H); 7.82 (s,1 H).

EXAMPLE 15

2-N-Cbz-6-benzyloxy-9-carboxymethylpurine (15)

2-Amino-6-benzyloxy-9-carboxymethylpurine is further protected withRappaport's Reagent following standard procedures and purified by silicagel column chromatography.

EXAMPLE 16

N2- (2S-Boc-aminocyclohex-1S-yl)-N-(adenin-1-ylacetyl)-glycine (16)

The title compound is synthesized as per the procedures of Example 4using the product from Example 15.

EXAMPLE 17

(1R,2R)-1-(N-t-butylcarbonylarino)-2-aminocyclohexane (17)

To a cooled solutionof (1R,2R)-(−)-trans-1,2-diamino-cyclohexane (5 ml;41.6 mmol) in CH₂Cl₂ (25 ml) was added a solution of di-t-butyldicarbonate (3.03 g, 13.9 mmol) in CH₂Cl₂ (25 ml) over a period of 30mins. The reaction mixture was stirred overnight at room temperature.Water (20 ml) and CH₂Cl₂ (25 ml) were added in order to dissolve theprecipitate. After separation, the organic phase was concentrated underreduced pressure and the residue dissolved in ether (25 ml) and water(25 ml). The mixture was acidified to pH 5 with HCl 4 N and thebis-protected diamine was extracted with ether (3×25 ml). The aqueousphase was adjusted to pH 10.5 with NaOH 2 N and extracted withethylacetate (6×30 ml). The organic phase was dried over sodium sulfate,filtered and evaporated under reduced pressure to yield thetitle-compound (2.171 g, 69.5% based on Boc₂O)

mp: 109-111° C.; NMR ¹H (dmso d6) ppm: 1.0-1.3 (m, 4 H, 2CH₂ cycl); 1.45(s, 9 H, t-Boc); 1.6 (m, 2 H, CH₂ cycl); 1.85 (m, 2 H, CH₂ cycl); 2.4(dt, 1 H, CHN); 2.9 (m, 1 H, CHN); 6.6 (m, 1 H, NH carbamate); NMR ¹³C(dmso d6) ppm: 155.6 (carbamate); 77.4 (t-Boc); 57.0, 53.8, 34.4, 32.2,25.8, 24.8 (C cycl); 28.4 (t-Boc).

EXAMPLE 18

N-(2R-Boc-aminocyclohex-1R-yl)-glycine, methyl ester (18)

To a cooled suspension of 17 (2 g, 9.35 mmol) and potassium carbonate(3.87 g, 28.05 mmol) in DMF (15 ml) was added a solution of methylbromoacetate (0.9 ml, 9.35 ml) in DMF (5 ml) over a period of 5 min.After one hour at 0° C. the salts were filtered and washed with DMF (15ml) and CH₂Cl₂ (15 ml). The filtrate was evaporated under reducedpressure and the residue was purified by chromatography on silica gel(eluent ethylacetate). Yield: 1.99 g (74%).

mp: 68-70° C.; NMR ¹H (dmso d6) ppm: 1.0-1.3 (m, 4 H, 2CH₂ cycl); 1.45(s, 9 H, t-Boc); 1.4-2.0 (m, SH, 2CH₂ cycl+NH); 2.3 (dt, 1 H, CHN); 3.1(m, 1 H, CHN); 3.4 (dt, 2 H, CH₂COO); 3.7 (s, 3 H, COOCH₃); 6.7 (m, 1 H,NH carbamate); NMR ₁₃C (dmso d6) ppm: 172.9 (ester); 155.5 (carbamate);77.5 (t-Boc); 59.7, 47.6 (CH₂COOCH₃); 53.7, 51.4, 32.1, 31.1, 24.6, 24.2(C cycl); 28.3 (t-Boc); MS FAB+: 287.0 (M+1).

EXAMPLE 19

N-(2R-Boc-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl)-glycine, methylester (19)

To a solution of 18 (1.5 g, 5.24 mmol), thymine acetic acid (0.96 g,5.22 mmol) and DhbtOH (0.85 g, 5.2 mmol) in DMF (20 ml) and CH₂Cl₂ (15ml) was added DCC (1.08 g, 5.24 mmol). After 4 hours at roomtemperature, DCU was filtered and washed with CH₂Cl₂ (100 ml). Thefiltrate was washed with NaHCO₃ 1 M (3×40 ml), KHSO₄ 1 M (2×40 ml) H₂O(40 ml). The organic phase was dried over sodium sulfate and filtered.Petroleum ether (100 ml) was added. After 48 h at 0° C., the titlecompound was collected by filtration. Yield: 1.77 g (74%)

mp: 205-207° C.; NMR ¹H (dmso d6) ppm: 1.2-2.0 (m, CH₂ cycl); 1.45 (s,t-Boc); 1.9 (CH₃ thymine); 3.7 (s, COOCH₃); 3.7 (dd, CH₂COO); 4.8 (dd,CH₂-T); 6.95 (m, NH carbamate); 7.2 (s, H-C═C-Me); 11.35 (s, NH imide);NMR ¹³C (dmso d6) ppm: 169.7, 167.1, 164.3, 154.9, 150.9 (C═O); 141.5,108.2 (C═C); 77.9 (t-Boc); 59.8, 47.5 (CH₂COOCH₃); 53.7, 51.4, 32.1,31.1, 24.6, 24.2 (C cycl); 28.2 (t-Boc); 11.9 (CH₃ thymine); MS FAB+:453.3 (M+1); 353.3 (M+1 - t-Boc).

EXAMPLE 20

N-(2R-Boc-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl) -glycine (20)

The monomer ester 19 (1.5 g, 3.3 mmol) was suspended in THF (15 ml) anda solution of LiOH 0.5 M (15 ml, 7.5 mmol) was added as well as water (5ml). After 45 min at room temperature water (30 ml) was added and themixture was washed with CH₂Cl₂ (3×30 ml). The aqueous phase wasacidified to pH 2.5-3 and extracted with ethylacetate (6×120 ml). Theorganic phase was dried over sodium sulfate and evaporated under reducedpressure to give the title compound. Yield: 1.38 g (95%).

NMR ¹H (dmso d6) ppm: 1.2-2.0 (m, CH₂ cycl) 1.45 (s, t-Boc); 1.9 (CH₃thymine); 3.9 (dd, CH₂COO); 4.8 (dd, CH₂-T); 6.95 (m, NH carbamate); 7.2(s, H-C═C-Me); 11.35 (s, NH imide); 12.4 (m, COOH); NMR ¹³C (dmso d6)ppm: 170.3, 166.8, 164.3, 155.0, 150.9 (C═O); 141.5, 108.1, (C═C); 77.9,28.2 (t-Boc); 59.7, 49.9, 47.9, 44.2, 32.1, 29.7, 24.4, 24.3, (C cycl+2CH₂); 11.9 (CH₃ thymine); MS FAB+: 439.2 (M+1); 339.1 (M+1 -t-Boc).

EXAMPLE 21

N-2-(2R-Boc-aminocyclohex-1R-yl)-N-(cytosin-1-ylacetyl)-glycine (21)

The title compound is synthesized as per the procedures of Examples 19and 20 using the product from Example 7.

EXAMPLE 22

N-2-(2R-Boc-aminocyclohex-1R-yl)-N-(adenin-1-ylacetyl) -glycine (8)

The title compound is synthesized as per the procedures of Examples 19and 20 using the product from Example 11.

EXAMPLE 23

N-2-(2R-Boc-aminocyclohex-1R-yl)-N-(adenin-1-ylacetyl)-glycine (16)

The title compound is synthesized as per the procedures of Examples 19and 20 using the product from Example 15.

EXAMPLE 24

Synthesis of PNA Oligomers by Solid Phase, General Procedure

The functionalized resin is measured out to typically provide 0.1-1.0millimoles of functionality, (functionalities attached to resins arecommercially available through various sources e.g. PeptidesInternational, Kentucky). This weight of resin is suspended in a 1:1(v:v) dichloromethane:dimethyl-formamide solution (30 mL/1 g of resin)and allowed to swell for a period of time if desired. The solvent isthen removed by filtration and the resin resuspended in trifluoroaceticacid (1 mL/1 gm of resin) and shaken for 3 minutes. The trifluoro-aceticacid is removed by filtration and this step is repeated once. The resinis washed three times with a solution of 1:1 (v:v)dichloromethane:dimethylformamide. The resulting resin is resuspended inpyridine solution (5 mL/1 gm of resin) and vacuum filtered to remove thepyridine. This step is repeated once. This is followed by resuspensionand filtration (designated “washing”) using 1:1 (v:v)dichloromethane:di-methylformamide solution (5 mL/1 g of resin) thiswashing step is repeated twice. The resin is suspended in 1:1 (v:v)pyridine:dimethylformamide and to this suspension is added the desiredPNA monomer (2-10 molar equivalents), TBTU (1.9-9.9 molar equivalents),and di-isopropylethylamine (5-20 molar equivalents) such that the finalconcentration of PNA monomer is 0.2 M. The suspension is shaken for15-60 minutes and the spent coupling solution is removed by filtration.The resin is washed with pyridine three times, and any unreacted aminesare capped using Rapoport's Reagent, 5 equivalents in DMF for 5 minutes.The resin is then washed three times with pyridine followed by threewashes with a solution of 1:1 (v:v) dichloro-methane:dimethylformamide(5 mL/1 gm of resin). At this point, the resin is ready for the nextcoupling reaction and this procedure is repeated until the desired PNAis assembled on the resin.

Specific Examples of Amino Ethyl Glycine (aeg-) PNAs and aeg-PNADerivatives Prepared by this General Method

Resin Employed aeg-PNA/aeg-PNA Derivative Prepared

Merrifield H₂N-GCAT-COOH (SEQ ID NO:1) Lys Substituted H₂N-GCAT-Lys-COOH(SEQ ID NO:2) Merrifield MBHA H₂N-GCAT-CONH₂ (SEQ ID NO:1) LysSubstituted H₂N-GCAT-Lys-CONH₂ (SEQ ID NO:2) MBHA

EXAMPLE 25

Oligomerization Using Chiral PNA Monomers

Solid phase synthesis of PNA oligomers having a chiral monomer wasperformed as per the procedures of Example 24. (4-methylbenzhydryl)amine(MBHA) resin was used with the initial loading at 0.1 meq/g usingHBTU/DIEA in DMF/pyridine as a coupling reagent. The free PNAs werereleased from the resin with TFMSA, purified by HPLC and characterizedby FAB-MS. [H-TTT TT_(cycRR)T TTT T-LysNH₂ (SEQ ID NO:3): M+1: calc.2860.9; found 2862.0; H-TTT TT_(cycSS)T TTT T-LysNH₂ (SEQ ID NO:4): M+1:calc. 2860.9; found 2861.7].

Specific Examples of Amino Ethyl Glycine (aeg-) PNAs Having a ChiralMonomer Incorporated Therein, Prepared by this General Method;

H-TTTTT*TTTTT-Lys-NH₂SEQ ID NO:3

H-TTTTT**TTTTT-Lys-NH₂SEQ ID NO:4

wherein T** denotes the SS monomer, N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine residue and a T* denotes the RR monomer,N-(2R-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl)-glycine residue.

Specific Examples of Amino Ethyl Glycine (aeg-) PNAs Having a ChiralMonomer Incorporated Therein, That are Prepared by this General Method;

H₂N-GC** T-COOH

H2N-GTA G*T CAC T-COOHSEQ ID NO:5

H₂N-CCA**GGC UCA GAT-COOHSEQ ID NO:6

H₂N-CT**G TCT CCA T**CC TCT T**CA CT-COOHSEQ ID NO:7

H₂N-TGG GA*G CC*A TAG CGA GCC-COOHSEQ ID NO:8

H₂N-TCT GA**G TAG CAG AGG AG**C TAA G-COOHSEQ ID NO:9

wherein ** denotes an SS monomer and a * denotes an RR monomer as shownabove except with mixed sequences.

EXAMPLE 26

Functionalization with Fluorescein

PNA oligomer SEQ ID NO:4 is synthesized as per the procedures of Example25. The oligomer is further functionalized with an N-Boc-aminohexanoicacid using the general procedure of Example 24. The Boc group is removedwith TFA as per the general procedures and the oligomer is furthercoupled with fluorescein-N-hydroxysuccinimide. The PNA oligomer iscleaved from, the resin and purified by reverse phase HPLC to give;

F-AHA-TTTTT**TTTTT-Lys-NH₂SEQ ID NO:4

wherein F represents fluorescein, AHA represents an aminohexanoic acidlinker and ** represents the SS monomer,N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine residue.

EXAMPLE 27

Functionalization with Rhodamine

As per the procedures of Example 26 rhodamine-N-hydroxysuccinimide isincorporated at the N terminus of PNA oligomer SEQ ID NO:4 to give;

R-ARA-TTTTT**TTTTT-Lys-NH₂SEQ ID NO:4

wherein R represents rhodamine, AHA represents an aminohexanoic acidlinker and ** represents the SS monomer,N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl) -glycine residue.

EXAMPLE 28

Thermal Stability Studies of Homopyrimidine PNA vs Homopyrimidine ChiralPNA Against DNA

The thermal stability of PNA/DNA and PNA***/DNA wherein PNA*** denotes aPNA oligomer containing one chiral (SS or RR) monomer was studied todetermine the effects of the chiral monomer on the Tm. When the SSisomer H-TTTTT-*TTTTT-Lys-NH₂ (SEQ ID NO:4; T** denoting the SS monomer,N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine residue) washybridized with the DNA 10mer A₁₀ (SEQ ID NO: 11), the Tm was comparableto that of the PNA(H-TTTTTTTTTT-Lys-NH₂; SEQ ID NO:10)/DNA complex.Results of hybridization of the DNA 10mer with the PNA's were comparedwith hybridazation of the PNA's with the same 10mer containing amismatch corresponding to a position occupied by a chiral monomer in thechiral PNA strand.

The results of the study show that the 10mer containing the SS isomershows equivalent binding affinity and comparable specificity to PNA.When the same thermal stability studies were performed on the RR isomerH-TTTTT*TTTTT-Lys-NH₂, (SEQ ID NO:6; T* denoting the RR monomer,N-(2R-aminocyclohex-1R-yl)-N-(thymin-1-ylacetyl) -glycine residue),there was seen poor binding affinity as well as poor specificity. Theresults of these studies are shown in Table 1.

TABLE 1 SEQ SEQ PNA ID NO: DNA ID NO: Tm H-TTTTT*TTTTT-LysNH₂ 6 (R,R) noDNA - (7° C.) H-TTTTT*TTTTT-LysNH₂ 6 (R,R) AAAAAAAAAA 11 (43° C.)H-TTTTT*TTTTT-LysNH₂ 6 (R,R) AAAACAAAAA 12 (40° C.)H-TTTTT**TTTTT-LysNH₂ 5 (S,S) no DNA - - H-TTTTT**TTTTT-LysNH₂ 5 (S,S)AAAAAAAAAA 11 (73° C.) H-TTTTT**TTTTT-LysNH₂ 5 (S,S) AAAACAAAAA 12 (60°C.) H-TTTTTTTTTT-LysNH₂ 10 AAAAAAAAAA 11 (76° C.) H-TTTTTTTTTT-LysNH₂ 10AAAACAAAAA 12 (63° C.)

The melting temperatures (Tm) of PNA/DNA hybrids were determined on aGilford Response apparatus. The conditions were 100 mM NaCl, 10 mM Naphosphate, 0.1 mM EDTA, pH=7. The following extinction cooeficients wereused: A, 10.8; T, 8.8; C, 7.3; mL/mmol-cm for both DNA and PNA. The Tmvalues were determined from the maximum of the first derivative of theplot of A₂₆₀ versus temperature.

The circular dichroism spectra of PNA oligomer SEQ ID NO:4 and PNAoligomer SEQ ID NO:3 are shown in FIG. 2. The conditions for the spectraare 5 mM phosphate buffer pH 7.0, 25 μM basetriplets (2:1 PNA T₁₀ [SEQID NO:10]:dA₁₀ [SEQ ID NO: 11]). Incubation was for 1 hour prior toanalysis. The spectra was run on a Jasco 720 instrument with a 2 nmbandpass, scan-rate 50 nm/min, lsec response time, with 6 averages ofeach spectrum. The temperature was maintained at 20° C. and thepathlength was 1 cm.

Homopyrimidine PNA₂/DNA triplexes are very similar in structure to thatof the DNA/DNA triplexes (see, Kim, S. K., et.al., J. Am. Chem. Soc.,1993, 115, 6477). As shown in the spectra PNA oligomer SEQ ID NO:4 andPNA oligomer SEQ ID NO:3 alone have a weak CD, due to chiral monomer andthe L-lysine residue. Upon hybridization to the complementary DNA thereis a strong CD response very similar to one that is obtained from anormal PNA/DNA complex. As expected from Tm experiments, the is (S,S)isomer SEQ ID NO:4, gave a stronger CD spectra than the (R,R) isomer SEQID NO:3. The two CD spectra of the complexes formed from the (R,R) and(S,S) isomers are not mirror images. Thus, the incorporation of onechiral PNA monomer did not disrupt the right handed helical structure ofthe PNA/DNA complex.

EXAMPLE 29

Thermal Stability Studies using a mixed sequence of PNA vs Chiral PNA

In a like manner to Example 28, the thermal stability of a chiral PNA ina mixed sequence was determined. The PNAs H-GT*AGAT*CACT*-Lys-N₂ (SEQ IDNO:13), GT**AGAT**CACT**-Lys-NH₂ (SEQ ID NO:14), and GTAGATCACT-Lys-NH₂(SEQ ID NO:15), wherein ** denotes an SS monomer and * denotes an RRmonomer, were synthesized in accordance with the general procedures ofExample 25. DNA oligonucleotides CATCTAGTG (SEQ ID NO: 16) and CATCTGGTG(SEQ ID NO: 17) were synthesized in accordance with methods known in theart. Both SEQ ID NO: 16 and SEQ ID NO: 17 were antiparallel to SEQ IDNO:15 with the exception that SEQ ID NO: 17 included a single basemismatch at position 6, substituting a guanine for the complementarybase pair adenine. The mismatch in SEQ ID NO: 17 ocurrs at a positionthat binds with a chiral position in the PNA oligomer, i.e. a chiralposition was incorporated at position 6 in each of SEQ ID NO: 13 and SEQID NO: 14. The chiral PNA oligomers (R,R, and S,S) and the unmodifiedPNA oligomer were hybridized with each of the two DNA oligomers.Relative binding specificity was measured using the methods andapparatus of Example 28.

Results show that the PNA lomer containing the PNA isomer shows (S,S)exhibited greater specificity than the unmodified PNA 10mer (23° C.T_(m) versus 10° C. T_(m), respectively). The PNA isomer (R,R) showedpoor binding affinity as well as poor specificity. The results areoutlined in Table 2.

TABLE 2 3′-CATCTAGTG-5′ 3′-CATCTGGTG-5′ (SEQ ID NO:16) (SEQ ID NO:17)OLIGOMER T_(m) T_(m) H-GT*AGAT*CACT*-Lys-NH₂ 34° C. no complex (SEQ IDNO:13) (R,R) detected GT**AGAT**CACT**-Lys-NH₂ 50° C. 27° C. (SEQ IDNO:14) (S,S) GTAGATCACT-Lys-NH₂ 54° C. 44° C. (SEQ ID NO:15)

EXAMPLE 30 PCR Assay for the Detection of a Single Point Mutation

A multitude of human genetic diseases result from a single base mutationin a specific gene. PCR in vitro analysis of a tissue or cell sampleusing a PNA oligomer is performed as per the procedures contained inOrum, H., et.al., Nucleic Acids Research, 1993, 21, 5332-5336. Samplesof interest are treated as per standard procedures to prepare genomicDNA for analysis. A PNA oligomer complementary to the wild type DNA inthe region of interest is synthesized having a chiral PNA monomer at theposition suspected of a point mutation. The sample is treated with anexcess of this PNA oligomer and an excess of the appropriate primersbracketing the region of interest. The mutant gene is amplified andcharacterized utilizing the procedures refereced above and standardmethods. If 100% of the target DNA is wild type no amplification willtake place. If mutant DNA is present the mutant DNA will be amplified.

EXAMPLE 31

Detection of mutant H-ras gene expression

Point mutations in the H-ras gene have been implicated in numerousaberrations of the Ras pathway. PNA oligomers are labeled aftersynthesis with fluorescein or other fluorescent tag. Labeled PNAoligomers are contacted with tissue or cell samples suspected ofabnormal ras expression under conditions in which specific hybridizationcan occur, and the sample is washed to remove unbound PNA oligomer.Label remaining in the sample indicates bound PNA oligomer and isquantitated using a fluorimeter, fluorescence microscope or otherroutine means.

Tissue or cell samples suspected of expressing a point mutation in theH-ras gene are incubated with a fluorescein-labeled PNA oligomer whichis targeted to the mutant codon 12, codon 13 or codon 61 of H-ras mRNA.An identical sample of cells or tissues is incubated with a secondlabeled PNA oligomer which is targeted to the same region of normalH-ras mRNA, under conditions in which specific hybridization can occur,and the sample is washed to remove unbound PNA oligomer. Label remainingin the sample indicates bound PNA oligomer and can be quantitated usinga fluorimeter or other routine means. The presence of mutant H-ras isindicated if the first sample binds labeled PNA oligomer and the secondsample does not bind fluorescent label.

Double labeling can also be used with PNA oligomers and methods of theinvention to specifically detect expression of mutant ras. A singletissue -sample is incubated with a rhodamine-labeled PNA oligomer whichis targeted to codon 12, codon 13 or codon 61 of mutant H-ras mRNA and afluorescein-labeled PNA oligomer which is targeted to the translationinitiation site of ras mRNA, under conditions in which specifichybridization can occur. The sample is washed to remove unbound PNAoligomer and labels are detected by and fluorimetry with appropriatefilters. The presence of mutant ras is indicated if the sample does notbind rhodamine-labeled PNA oligomer but does retain the fluoresceinlabel.

EXAMPLE 32

Detection of mutant β-amyloid precursor protein gene expression (NAPP)

Point mutations in the gene encoding β-amyloid have been implicated infamilial Alzheimer's disease (FAD). PNA oligomers are labeled aftersynthesis with fluorescein or other fluorescent tag. Labeled PNAoligomers are contacted with tissue or cell samples suspected ofabnormal βAPP expression under conditions in which specifichybridization can occur, and the sample is washed to remove unbound PNAoligomers. Label remaining in the sample indicates bound oligonucleotideand is quantitated using a fluorimeter, fluorescence microscope or otherroutine means.

Tissue or cell samples suspected of expressing a point mutation in theβAPP gene are incubated with a fluorescein-labeled PNA oligomer which istargeted to the mutant codon 717, codon 670 or codon 671 of βAPP mRNA.An identical sample of cells or tissues is incubated with a secondlabeled PNA oligomer which is targeted to the same region of normal βAPPmRNA, under conditions in which specific hybridization can occur, andthe sample is washed to remove unbound PNA oligomer. Label remaining inthe sample indicates bound PNA oligomer and can be quantitated using afluorimeter or other routine means. The presence of mutant βAPP isindicated if the first sample binds labeled PNA oligomer and the secondsample does not bind fluorescent label.

Double labeling can also be used with PNA oligomers and methods of theinvention to specifically detect expression of mutant βAPP. A singletissue sample is incubated with a rhodamine-labeled PNA oligomer whichis targeted to codon 717, codon 670 or codon 671 of mutant βAPP mRNA anda fluorescein-labeled PNA oligomer which is targeted to the translationinitiation site of mutant βAPP MRNA, under conditions in which specifichybridization can occur. The sample is washed to remove unbound PNAoligomer and labels are detected by and fluorimetry with appropriatefilters. The presence of mutant βAPP is indicated if the sample does notbind rhodamine-labeled PNA oligomer but does retain the fluoresceinlabel.

17 4 amino acid single unknown peptide unknown Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= Modified-site /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=Modified-site /note= N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site /label= Modified-site /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 1 Xaa Xaa Xaa Xaa 5 aminoacid single unknown peptide unknown Modified-site /label= MODIFIED-SITE/note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site/label= Modified-site /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)-glycine Modified-site /label= Modified-site /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=Modified-site /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 2 XaaXaa Xaa Xaa Lys 5 11 amino acid single unknown peptide unknownModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(2R-aminocyclohex-1R-yl)-N-(thymin- 1-ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 3 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 11 amino acid singleunknown peptide unknown Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(2S-aminocyclohex-1S-yl)-N-(thymin- 1-ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 4 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 9 amino acid singleunknown peptide unknown Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(2R-aminocyclohex-1R-yl)-N-(guanin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 5 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa 1 5 12 amino acid single unknown peptide unknownModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(2S-aminocyclohex-1S-yl)-N-(adenin- 1-ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(uracil-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 11 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 12 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 6 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 20 amino acid singleunknown peptide unknown Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note= N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine Modified-site 11 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 12 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site 13 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site 14 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site 15 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site 16 /label=MODIFIED-SITE /note= N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine Modified-site 17 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 18 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site 19 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 20 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 7 XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 XaaXaa Xaa Xaa Xaa 20 18 amino acid single unknown peptide unknownModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(2R-aminocyclohex-1R-yl)-N-(adenin- 1-ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(2R-aminocyclohex-1R-yl)-N-(cytosin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site 11 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 12 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 13 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 14 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 15 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 16 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 17 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 18 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine 8Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15Xaa Xaa Xaa 22 amino acid single unknown peptide unknown Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(2S-aminocyclohex-1S-yl)-N-(adenin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 10 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 11 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site 12 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site 13 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site 14 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site 15 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site 16 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 17 /label=MODIFIED-SITE /note= N-(2S-aminocyclohex-1S-yl)-N-(guanin-1-ylacetyl)-glycine Modified-site 18 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 19 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site 20 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site 21 /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site 22 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine 9 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa XaaXaa 20 11 amino acid single unknown peptide unknown Modified-site/label= MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note= N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site 10 /label= MODIFIED-SITE /note=N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine 10 Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa Xaa Xaa Lys 1 5 10 10 nucleic acid single linear unknown 11AAAAAAAAAA 10 10 nucleic acid single linear unknown 12 AAAACAAAAA 10 11amino acid single unknown peptide unknown Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(2R-aminocyclohex-1R-yl)-N-(thymin-1- ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note N-(aminoethyl)-N-(adenin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(2R-aminocyclohex-1R-yl)-N-(thymin-1- ylacetyl)-glycine Modified-site/label= MODIFIED-SITE /note N-(aminoethyl)-N-(cytosin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycineModified-site 10 /label= MODIFIED-SITE /noteN-(2R-aminocyclohex-1R-yl)-N-(thymin-1- ylacetyl)-glycine 13 Xaa Xaa XaaXaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 11 amino acid single unknownpeptide unknown Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(guanin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note N-(2S-aminocyclohex-1S-yl)-N-(thymin-1-ylacetyl)-glycine 14 Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 1011 amino acid single unknown peptide unknown Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /note N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycine Modified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(guanin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(adenin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(thymin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site /label=MODIFIED-SITE /note N-(aminoethyl)-N-(adenin-1-ylacetyl)- glycineModified-site /label= MODIFIED-SITE /noteN-(aminoethyl)-N-(cytosin-1-ylacetyl)- glycine Modified-site 10 /label=MODIFIED-SITE /note N-(aminoethyl)-N-(thymin-1- ylacetyl)- glycine 15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Lys 1 5 10 9 nucleic acid singlelinear unknown 16 CATCTAGTG 9 9 nucleic acid single linear unknown 17CATCTGGTG 9

What is claimed is:
 1. A peptide nucleic acid oligomer complementary toa target molecule, said oligomer being composed of peptide nucleic acidmonomers having a (2-aminoethyl)glycine backbone, wherein the Cα and Cβcarbons of each of said monomers form part of an alicyclic structurewhich renders said Cα and Cβ carbons chiral.
 2. A peptide nucleic acidoligomer complementary to a target molecule, said oligomer beingcomposed of: (a) peptide nucleic acid monomers having a nonchiral(2-aminoethyl)glycine backbone; and (b) chiral peptide nucleic acidmonomers having a (2-aminoethyl)glycine backbone, wherein the Cα and Cβcarbons of each of said chiral PNA monomers form part of an alicyclicstructure which renders said Cα and Cβ carbons chiral.
 3. The oligomerof claim 2 wherein the chiral peptide nucleic acid monomers have thestructure:

wherein: B is a naturally occurring or non-naturally occurringnucleobase; n is 0, 1, 2, or 3; and at least one of Cα or Cβ is in the Sconfiguration.
 4. The oligomer of claim 3 wherein n is 2, and B isadenine, cytosine, guanine, thymine, or uracil.
 5. The oligomer of claim3 wherein B is adenine, cytosine, guanine, thymine, or uracil.